PROJECT SUMMARY/ABSTRACT Clostridium difficile-associated disease (CDAD) is an increasing problem in Veterans Hospitals and other hospitals throughout North America. Recent multi-hospital outbreaks in Canada and in the U.S. have been linked to the emergence of a specific C. difficile toxin variant, referred to as BI/NAP1/027 which may explain, at least in part, the overall increase in national rates of CDAD. The toxin variant BI/NAP1/027 strains are characterized by increased production of toxin A and B (the major known virulence factors of C. difficile), the presence of an additional toxin, referred to as binary toxin CDT, as well as increased resistance to fluoroquinolone antibiotics. Because of their association with more severe disease in these hospital outbreaks, the BI/NAP1/027 strains have been referred to as hypervirulent. Determining the contribution of the various toxins and other factors to hypervirulence of the BI strains will not only improve the understanding of why these strains have become the dominant epidemic strains in North America, but will also help elucidate the fundamental molecular steps in the pathogenesis of CDAD. Historically, toxin A has been implicated as the primary virulence determinant of C. difficile because of its potent enterotoxic activity and the ability of toxin A alone to produce disease in animal models. However, clinical observations on the naturally-occurring toxin A- negative, B-positive (A-/B+) variant strains, and our work demonstrating virulence of A-/B+ strains in the hamster model, in addition to other recent evidence, suggest that toxin B, not A, is the essential virulence factor in C. difficile. We hypothesize that toxin B, not toxin A is the essential virulence factor of C. difficile, including epidemic BI strains. We also hypothesize that the frequency of a particular strain in our hospital will be determined in part by the level of toxin B production by that strain. The specific objectives of this proposal are: Objective 1: To prospectively screen C. difficile clinical isolates to determine the frequency of specific strains, correlate toxin production in vitro with frequency, and to obtain candidate hypervirulent BI isolates for construction of mutants. Objective 2: To construct independent mutants of toxin B and toxin A in a hypervirulent BI strain. Objective 3: To conduct in vitro analysis of the BI-derived toxin B and toxin A mutants for toxin production and for pathogenicity locus (PaLoc) gene expression. Objective 4: To test the independent toxin B and toxin A mutants in the hamster model to determine if virulence is abrogated. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Potential impact on Veterans Health Care: CDAD remains the major cause of hospital-acquired infectious diarrhea and VA hospitals have been particularly affected because the risk factors for CDAD, advanced age, prolonged hospital stay and frequent antibiotic exposure are characteristics of VA Hospital patients. An improved understanding of the pathogenesis of CDAD, should lead to new treatments, diagnosis, and strategies to interrupt the ongoing epidemic of CDAD affecting VA and non-VA hospitalized patients.